The versatile plant acyltransferase (VPAT) family is a recently identified protein family consisting of acyltransferases involved in secondary metabolism in plants along with numerous homologues with as yet unidentified biochemical functions. Malonyl-CoA:anthocyanin 5-O-glucoside-6' "-O-malonyltransferase of Salvia splendens flowers (Ss5MaT1) is a member of this family that catalyzes the regiospecific transfer of the malonyl group from malonyl-CoA to the 6' "-hydroxyl group of the 5-glycosyl moiety of anthocyanins. To elucidate the mechanism and functional amino acid residues of VPAT family enzymes, steady-state kinetic analyses and site-directed mutagenesis of Ss5MaT1 guided by sequence comparison studies were carried out. On the basis of the results of product and dead-end inhibition studies as well as sequence comparison studies, the kinetic mechanism of Ss5MaT1 could be most consistently described in terms of a ternary complex mechanism in which both substrates and the enzyme form a complex before catalysis can occur, as in the case of chloramphenicol O-acetyltransferase (CAT) and histone acetyltransferase (HAT). Eight polar or ionizable amino acid residues that are invariant among 12 VPAT family enzymes were replaced by alanine, and the mutant enzymes were kinetically characterized. A significant diminution of the k(cat) value was observed with the substitution of His167 (relative k(cat), 0.02%) and Asp390 (<0.01%), strongly suggesting that His167 and Asp390 are very important for catalytic activity. The log k(cat) versus pH plots of the Ss5MaT1-catalyzed malonyl transfer suggested that a deprotonated active site group of pK(a) = 7.0 +/- 0.1 may be involved in the catalytic steps of the "substrate to product" conversion in the ternary enzyme-substrate complex. Taking these lines of evidence together with the suggested similarity of the kinetic and catalytic mechanisms of Ss5MaT1 to those of CAT and HAT, the following Ss5MaT1 mechanism based on general acid/base catalysis was proposed: in the ternary complex, a general base deprotonates the 6' "-hydroxyl group of the anthocyanin substrate, thereby promoting a nucleophilic attack on the carbonyl of the thioester of malonyl-CoA; His167 and Asp390 appear to be involved in the general acid/base mechanism of Ss5MaT1.